Staurosporine solution

The colorimetric MTT assay was used to evaluate the metabolic activity as indicator of the activation, proliferation and cytotoxicity [24 (link)]. Calu-3 cells were incubated with the inhibitors in a mono- or double-treatment using the indicated concentrations for 48 h. Staurosporine solution (1 µM) (Sigma) served as a positive control for cytotoxic effects. Following 48 h of treatment, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (1 mg/mL) (Sigma) was added to the cells for 3 h. The supernatant was aspirated, DMSO was added to the cells for 10 min and the OD₅₆₂ was subsequently measured using an Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA) to evaluate the cell viability according to the manufacturer’s protocol (Sigma).

Methyltrienolone (R1881, Perkin Elmer, cat. no. NLP005005MG) and paclitaxel (LC Laboratories, cat. no. P-9600) were pre-dissolved in DMSO to obtain 10 and 20 mM stock solutions, respectively. Staurosporine solution, 1 mM in DMSO, was purchased from Sigma-Aldrich (cat. no. S6942). For cell treatment, working solutions of a 10x concentration of the desired end concentration were prepared using media. In the course of the experiment, cells in 90 μl media were treated with 10 μl working solution to achieve a 100 μl total volume of 50 nM paclitaxel or 250 nM staurosporine for each well.

Tryptic soya broth and agar were purchased from Hi-Media Laboratories. Lysozyme, Sodium dodecyl sulfate (SDS), absolute ethanol, necrostatin-1, XTT sodium salt [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide], MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], menadione, staurosporine solution, ionomycin, glutaraldehyde, paraformaldehyde, and Triton X-100 were procured from Sigma-Aldrich. Tris-buffer, 1-octanol, HCl, and chloroform, were obtained from Merck-Millipore. RPMI medium 1640, HEPES buffer, penicillin-streptomycin solution (Pen-Strep), fetal bovine serum (FBS), and TRIzol Reagent were purchased from Gibco-Invitrogen. L-selenomethionine was purchased from Calbiochem. ApoTox-Glo Triplex assay kit for measuring Caspase3/7 levels and CytoTox-ONE™ Homogeneous Membrane Integrity Assay kit for LDH release assay were purchased from Promega and the manufacturer's protocol was followed. Verso cDNA Synthesis Kit and DyNAmo ColorFlash SYBR Green qPCR kit were obtained from ThermoFisher Scientific. Limulus Amebocyte Lysate (LAL) reagent and standard endotoxin were purchased from Lonza. All plastic ware for cell culture was purchased from Nunc. Type I Millipore water was used in all the experiments.